RIP140 regulation of diabetes

ABSTRACT

Inhibition of RIP140 increases glucose transport. Compounds that inhibit RIP140 expression or activity are useful for treating disorders associated with aberrant glucose transport (e.g., diabetes), treating obesity, increasing metabolism (e.g., fatty acid metabolism), and increasing brown fat.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. patentapplication Ser. No. 11/075,646, filed on Mar. 7, 2005, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/550,677,filed on Mar. 5, 2004. These prior applications are herein incorporatedby reference in their entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant no. DK030648awarded by the National Institutes of Health.

The government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to molecular biology, cell biology, cellularmetabolism, and diabetes.

BACKGROUND

Insulin stimulates glucose transport in muscle and fat. One of the mostcritical pathways that insulin activates is the rapid uptake of glucosefrom the circulation in both muscle and adipose tissue. Most ofinsulin's effect on glucose uptake in these tissues is dependent on theinsulin-sensitive glucose transporter, GLUT4 (reviewed in Czech andCorvera, J. Biol. Chem., 274:1865-1868, 1999; Martin et al., CellBiochem. Biophys., 30:89-113, 1999; Elmendorf et al., Exp. Cell Res.,253:55-62, 1999). The mechanism of insulin action is impaired indiabetes, leading to less glucose transport into muscle and fat. This isthought to be a primary defect in type II diabetes. Potentiating insulinaction has a beneficial effect on type II diabetes. This is believed tobe the mechanism of action of the drug Rezulin (troglitazone).

Type II diabetes mellitus (non-insulin-dependent diabetes) is a group ofdisorders characterized by hyperglycemia that can involve an impairedinsulin secretory response to glucose and insulin resistance. One effectobserved in type II diabetes is a decreased effectiveness of insulin instimulating glucose uptake by skeletal muscle. Type II diabetes accountsfor about 85-90% of all diabetes cases.

RIP140 (receptor interacting protein 140, also known as NRIP1, forNuclear Receptor-interacting Protein 1) is a corepressor that caninhibit the transcriptional activity of a number of nuclear receptors.

SUMMARY

The present invention relates to findings regarding the role of RIP140in glucose transport.

Accordingly, the invention relates to methods of increasing glucosetransport in a cell by inhibiting RIP140 expression or activity. In somecases, RIP140 expression is inhibited by a mechanism that involves RNAinhibition (RNAi), e.g., an siRNA or other mechanism related to the useof a nucleic acid (e.g., an antisense nucleic acid that is targeted toRIP140).

Accordingly, in one aspect, the invention features a method forincreasing glucose transport in a cell by providing a cell andcontacting the cell with an agent that inhibits expression or activityof a RIP140 polypeptide, thereby increasing glucose transport in thecell. Also featured is a method for decreasing glucose transport byproviding a cell and contacting the cell with an agent that increasesexpression or activity of a RIP 140 polypeptide, thereby increasingglucose transport in the cell.

The agent can include a polynucleotide, a polypeptide, a smallnon-nucleic acid organic molecule, a small inorganic molecule, or anantibody, e.g., a small inhibitory RNA (siRNA) (e.g., including asequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:4) an antisense oligonucleotide, an inhibitory RNA, or a ribozyme.The cell is contacted in vitro or in vitro.

In some embodiments, the agent inhibits RIP140-mediated suppression ofexpression of a gene. In some embodiments, the agent decreases bindingof RIP140 polypeptide to a second polypeptide. In some embodiments, thesecond polypeptide is a Peroxisome Proliferator-Activated Receptor(PPAR), e.g., PPAR alpha, PPAR delta, or PPAR gamma.

Also provided herein is a method for increasing insulin-stimulatedglucose uptake in a subject (e.g., a human) that is at risk for orsuffering from a disorder related to glucose metabolism (e.g., diabetesor obesity), by administering to the subject an agent that decreasesexpression or activity of a RIP140 polypeptide in an amount sufficientto modulate glucose metabolism in a cell of the subject, therebyincreasing insulin-stimulated glucose uptake in the subject.

The disorder can be type I diabetes, type II diabetes. In someembodiments, the agent is an siRNA (e.g., an siRNA comprising SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4)

Also provided herein is a method for identifying a candidate agent thatmodulates expression or activity of a RIP140 polypeptide. The methodincludes (a) providing a sample comprising a RIP140 polypeptide or anucleic acid encoding the polypeptide; (b) contacting the sample with atest compound under conditions in which the polypeptide is active, thenucleic acid is expressed, or both; (c) evaluating expression oractivity of the RIP140 polypeptide in the sample; and (d) comparing theexpression or activity of the RIP140 polypeptide of (c) to expression oractivity of the RIP140 polypeptide in a control sample lacking the testcompound, wherein a change in RIP140 polypeptide expression or activityindicates that the test compound is a candidate agent that can modulatethe expression or activity of the RIP140 polypeptide.

The sample can include a cell, e.g., an adipocyte. In other embodiments,the sample is a cell-free sample. In various embodiments, the evaluatingincludes performing a cell-free assay.

The evaluating can include determining whether glucose transport ismodulated in the presence of the test compound, e.g., by determiningglucose uptake.

The test compound can include a polynucleotide, a polypeptide, a smallnon-nucleic acid organic molecule, a small inorganic molecule, or anantibody, e.g., an antisense oligonucleotide, an inhibitory RNA, or aribozyme.

In some embodiments, modulation of glucose transport is evaluated usingan antibody. Glucose transport can be increased or decreased in thepresence of the test compound.

In some embodiments, an activity of RIP140 is evaluated, e.g., bydetermining the level of interaction of RIP140 with a secondpolypeptide, e.g., a PPAR, e.g., PPARalpha, PPARdelta, or PPARgamma.

The method can further include testing the candidate agent in an animalmodel, e.g., an animal model of obesity or diabetes, e.g, by evaluatingRIP140 RNA levels, and/or by evaluating one or more of insulin levels orglucose levels. The method can further include, e.g., optimizing theagent and formulating the agent for pharmaceutical use.

In some embodiments, RIP140-mediated modulation of gene expression isevaluated, e.g. RIP140-mediated suppression of gene expression, orRIP140-mediated enhancement of gene expression. A change in RIP140activity can be evaluated by determining a change in the level ofexpression of a second gene, relative to a control.

Also provided herein is a method of increasing the amount of brown fatin a subject by administering an amount of a RIP140 inhibitor sufficientto decrease the level of expression or activity of RIP140 in a white fatcell to the subject. Increasing brown fat can increase the level ofmetabolism in the subject and thereby decrease obesity.

Also provided herein is method for identifying a candidate agent thatmodulates expression or activity of a polypeptide selected from thegroup consisting of C/EBP beta, C/EBP zeta, GA repeat binding proteinalpha, LXR beta, EAR2, Nur77, nuclear receptor binding factor 1, nuclearreceptor interacting protein 3, PPAR alpha, PPAR binding protein, PPARgamma, and coactivator 1 beta, the method including (a) providing asample comprising the polypeptide or a nucleic acid encoding thepolypeptide; (b) contacting the sample with a test compound underconditions in which the polypeptide is active, the nucleic acid isexpressed, or both; (c) evaluating expression or activity of thepolypeptide in the sample; and (d) comparing the expression or activityof the polypeptide of (c) to expression or activity of the polypeptidein a control sample lacking the test compound, wherein a change inpolypeptide expression or activity indicates that the test compound is acandidate agent that can modulate the expression or activity of thepolypeptide.

Also provided herein is a method for identifying a candidate agent thatmodulates expression or activity of a polypeptide selected from groupconsisting of C/EBP delta, C/EBP gamma, Rev-ErbA alpha, LXR alpha,COUP/TFII beta, progesterone receptor membrane component 1, progesteronereceptor membrane component 2, and retinoid receptor beta, by (a)providing a sample comprising the polypeptide or a nucleic acid encodingthe polypeptide; (b) contacting the sample with a test compound underconditions in which the polypeptide is active, the nucleic acid isexpressed, or both; (c) evaluating expression or activity of thepolypeptide in the sample; and (d) comparing the expression or activityof the polypeptide of (c) to expression or activity of the polypeptidein a control sample lacking the test compound, wherein a change inpolypeptide expression or activity indicates that the test compound is acandidate agent that can modulate the expression or activity of thepolypeptide.

In general, the methods in which cellular functions are assayed, thecell used in the assay can conduct glucose transport (e.g., is anadipose cell, a muscle cell, or a liver cell). Thus, in certain methods,a compound that can bind RIP140 is assayed in a cell that can conductglucose transport (and optionally, is insulin sensitive), contacting athe cell with a compound that can bind to RIP140, and determiningwhether the cell increases a cellular function associated with glucosetransport or increased fatty acid metabolism.

The invention also features a method of identifying a candidate compoundfor increasing glucose transport in a cell. The method includesproviding a sample comprising a Peroxisome Proliferator-ActivatedReceptor (PPAR), contacting the sample with a RIP140 and a testcompound, determining the level of interaction between the PPAR(PPARalpha, PPARdelta, or PPARgamma) and RIP140, such that a decrease inthe level of interaction between the PPAR and RIP140 in the presence ofthe test compound compared to a control that does not comprise the testcompound indicates that the compound is a candidate compound forincreasing glucose transport in a cell.

In another embodiment, the invention relates to a method of increasingthe amount of brown fat in a subject. The method includes administeringan amount of a RIP140 inhibitor sufficient to decrease the level ofexpression or activity of RIP140 in a white fat cell to the subject. Insome embodiments, the RIP inhibitor is an siRNA or other nucleic acidthat functions in the RNAi pathway.

By “specifically binds” is meant a molecule that binds to a particularentity, e.g., a RIP140 polypeptide in a sample, but which does notsubstantially recognize or bind to other molecules in the sample, e.g.,a biological sample, which includes the particular entity, e.g., aRIP140 polypeptide.

A “polypeptide” is a chain of amino acids regardless of length orpost-translational modifications. Thus, the terms polypeptides, protein,and peptide are used interchangeably. As used herein, the term “RIP140”means a RIP140 polypeptide.

An animal or human, is “at risk for” or “predisposed to” developing acondition such as a glucose transport-related disorder (e.g., type IIdiabetes) if there is an increased probability that it will develop thecondition compared to a population (e.g., the general population, anage-matched population, a population of the same sex). The increasedprobability can be due to one or a combination of factors including thepresence of specific alleles/mutations of a gene or exposure to aparticular environment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a list of sequences identified as having significant homologyto RIP140.

FIG. 2 is a bar graph illustrating the results of experiments in whichcells were transfected with siRNA targeting RIP140, PTEN, Akt, orscrambled and the amount of Akt phosphorylation determined in thepresence or absence of insulin.

FIG. 3 is a bar graph illustrating the results of experiments in whichcells were transfected with siRNA targeting RIP140, PTEN, or scrambled(control) and deoxyglucose transport assayed in the presence or absenceof insulin.

FIG. 4 is a bar graph illustrating the results of experiments in whichcells were transfected with siRNA targeting Akt2, PTEN, Nrip1, Smpd1,TUG long, Anxa8, or scrambled (control) and deoxyglucose transportassayed in the presence or absence of insulin.

FIG. 5 is a chart indicating the results of experiments detecting thelevel of expression of the listed expressed sequences in micecharacterized by having different features of glucose metabolism.

FIG. 6 is a set of photographs of Western blots depicting levels ofGLUT4, adiponectin, GLUT1, and actin polypeptides in day 4 3T3-L1adipocytes transfected with scrambled siRNA (Scr) or RIP140 siRNA.

FIG. 7 is a bar graph depicting the results of assays to determineRIP140 depletion in day 4 or day 8 3T3-L1 adipocytes treated withscrambled siRNA or RIP140 siRNA. Levels were determined by real-time PCRafter three days of expression.

FIG. 8 is a bar graph depicting the results of assays to determineRIP140 regulation of glucose in 3T3-L1 adipocytes transfected withscrambled or RIP140 siRNA after 4 or 8 days of differentiation. Levelsof 2-deoxyglucose depletion was measured in these assays. The graphshows the average and standard error of five or more independentexperiments. *p<0.05 compared to scrambled by student's t-test.

FIG. 9A is a set of photographs of Western blots depicting levels ofGLUT4 and PPARγ protein in day 8 3T3-L1 adipocytes transfected withscrambled, RIP140, PPARγ, or RIP140+PPARγ siRNA. Expression was measured72 hours after transfection.

FIG. 9B is a bar graph depicting the results of assays to determineRIP140 regulation of glucose in 3T3-L1 adipocytes transfected withscrambled, RIP140, PPARγ, or RIP140+PPARγ siRNA after 8 days ofdifferentiation. Levels of 2-deoxyglucose depletion was measured inthese assays. The graph shows the average and standard error of fiveindependent experiments. *p<0.05 compared to scrambled by student'st-test.

FIG. 10 is a diagram containing graphs of results of assays in whichexpression profiles of genes involved in glycolysis, fatty acidoxidation (FAO), the TCA cycle, and oxidative phosphorylation wereexamined by Affymetrix gene chip analysis.

FIG. 11 is a table listing the Affymetrix probe, GenBank® AccessionNumber, gene name, gene symbol, and fold change in genes in response toRIP140 depletion, as measured by Affymetrix gene chip analysis. Genechip analysis was performed on day 8 3T3-L1 adipocytes electroporatedwith scrambled siRNA or RIP140 siRNA. Fold change indicates thedifference in expression between scrambled- and RIP140 siRNA-transfectedcells.

FIG. 12A is a representation of a human RIP140 nucleotide sequence (SEQID NO: 5).

FIG. 12B is a representation of a human RIP140 amino acid sequence (SEQID NO: 6).

FIG. 13A is a representation of a murine RIP140 nucleotide sequence (SEQID NO: 7).

FIG. 13B is a representation of a murine RIP140 amino acid sequence (SEQID NO: 8).

DETAILED DESCRIPTION

It has been found that RIP140 plays a role in glucose transport and thedecreasing the level of RIP140 expression increases glucose transport.Thus, compounds that decrease RIP140 expression or activity are usefulfor increasing glucose transport and can be used as therapeutics fortreating disorders in which it is desirable to increase glucosetransport, for example, in diabetes. Decreasing RIP140 expression oractivity is also useful for increasing the amount of brown fat, therebyincreasing metabolism and thus promoting weight loss. Compounds that candecrease RIP expression or activity are useful for treating conditionsor disorders associated with an undesirable amount of white fat, e.g.,obesity or obesity associate with type II diabetes. Furthermore, RIP140interacts with several different PPARs. Compounds that decrease theinteraction between RIP140 and a ligand such as a PPAR (PeroxisomeProliferator-Activated Receptor; e.g., PPARalpha, PPARdelta, andPPARgamma) are useful for treating disorders associated with RIP140activity, e.g., diabetes or obesity. Furthermore, compounds that inhibitRIP140 expression or activity not only are useful for increasing glucoseuptake in a cell, but are also useful for increasing insulin sensitivityof a cell, and are generally useful for activating transcription throughPPARs and enhancing energy expenditure and cellular metabolism.

RIP140 and PPAR

RIP140 is a nuclear protein containing approximately 1158 amino acids,with a size of approximately 128 kDa. RIP140 binds to nuclear receptorsvia LXXLL motifs, wherein L is leucine and X is any amino acid (Heery etal., Nature, 387(6634):733-6, 1997). Ten LXXLL motifs are found in theRIP140 sequence. RIP140 also interacts with histone deacetylases andwith C-terminal binding protein (CTBP) via a PXDLS motif found in theRIP140 sequence.

A human RIP140 nucleotide sequence is listed in GenBank® under AccessionNo. NM_(—)003489. The corresponding human amino acid sequence is foundunder Accession No. NP_(—)003480. The nucleotide sequence of thechromosomal region containing the entire human RIP140 gene can be foundin GenBank® under Accession No. AF248484. A murine RIP140 nucleotidesequence can be found in GenBank® under Accession No. NM_(—)173440. Thecorresponding murine amino acid sequence is found under Accession No.NP_(—)775616. RIP140 is highly conserved between vertebrate species.

A number of RIP140 homologs are known in the art. A partial list of suchsequences is provided in FIG. 1. Inhibition of expression of a RIP140 ina cell that normally conducts glucose transport in response tostimulation by insulin (e.g., a fat cell) results in increased glucosetransport. A biologically active RIP140 or fragment thereof includessequences that can be transfected into a RIP140−/− cell and restoreRIP140 activity.

In some embodiments, RIP140 activity can be determined by examininglevels of RIP140 binding to PPARs. PPAR sequences are known in the art,for example see Genbank® accession nos. NP005027 (PPARalpha), Q03181(PPARdelta), P37231 (PPARgamma).

Screening Assays

The methods described herein include methods (also referred to herein as“screening assays”) for identifying modulators, i.e., test compounds oragents, of RIP140 expression or RIP140 activity. Such test compoundsinclude, e.g., polypeptides, peptides, peptidomimetics, peptoids, smallinorganic molecules, small non-nucleic acid organic molecules, nucleicacids (e.g., anti-sense nucleic acids, siRNA, oligonucleotides,synthetic oligonucleotides), carbohydrates, or other agents that bind toRIP140 proteins, have a stimulatory or inhibitory effect on, forexample, RIP140 expression or RIP140 activity, or have a stimulatory orinhibitory effect on, for example, the expression or activity of aRIP140 substrate. Compounds thus identified can be used to modulate theactivity of target gene products (e.g., RIP140 genes) in a therapeuticprotocol, to elaborate the biological function of a RIP140, or toidentify compounds that disrupt RIP140 interactions (e.g., with a PPARsuch as PPARalpha, PPARdelta, or PPARgamma).

In general, screening assays involve assaying the effect of a test agenton expression or activity of a RIP140 nucleic acid or polypeptide in atest sample (i.e., a sample containing the RIP140 nucleic acid orpolypeptide). Expression or activity in the presence of the testcompound or agent is compared to expression or activity in a controlsample (i.e., a sample containing a RIP140 polypeptide that wasincubated under the same conditions, but without the test compound). Achange in the expression or activity of the RIP140 nucleic acid orpolypeptide in the test sample compared to the control indicates thatthe test agent or compound modulates expression or activity of theRIP140 nucleic acid or polypeptide and is a candidate agent.

Compounds can be tested for their ability to modulate one or more RIP140mediated activities. For example, compounds that inhibit RIP140 activityresult in at least one of increased insulin sensitivity, increasedglucose transport, increased energy expenditure, increased metabolism,or increased fatty acid oxidation. Methods of assaying a compound forsuch activities are known in the art. In some cases, a compound istested for it's ability to directly affect RIP140 expression or bindingto a RIP140 ligand (e.g., by decreasing the amount of RIP140 RNA in acell, decreasing the amount of RIP140 protein in a cell, or decreasingthe repressor-associated binding of RIP140) and tested for its abilityto modulate a metabolic effect associated with RIP140 (e.g., increasedinsulin sensitivity, increased glucose transport, increased energyexpenditure, increased metabolism, or increased fatty acid oxidation).

In one embodiment, assays are provided for screening candidate or testmolecules that are substrates of a RIP140 polypeptide or a biologicallyactive portion thereof in a cell that is insulin sensitive (e.g., a cellthat can increase glucose transport in response to insulin). In anotherembodiment, the assays are for screening candidate or test compoundsthat bind to a RIP140 or modulate the activity of a RIP140 or abiologically active portion thereof. Such compounds include those thatdisrupt the interaction between RIP140 and a PPAR (e.g., PPARalpha,PPARdelta, or PPARgamma).

The test compounds used in the methods can be obtained using any of thenumerous approaches in the art including combinatorial library methods,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; e.g., Zuckermann et al., J. Med.Chem., 37:2678-2685, 1994); spatially addressable parallel solid phaseor solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam, Anticancer DrugDes., 12:145, 1997).

Examples of methods for the synthesis of molecular libraries can befound in the literature, for example in: DeWitt et al., Proc. Natl.Acad. Sci. USA, 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA,91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho etal., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl.33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl., 33:2061,1994; and Gallop et al., J. Med. Chem., 37:1233, 1994.

Libraries of compounds may be presented in solution (e.g., Houghten,Bio/Techniques, 13:412-421, 1992), or on beads (Lam, Nature, 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (U.S. Pat. No.5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409),plasmids (Cull et al., Proc. Natl. Acad. Sci. USA, 89:1865-1869, 1992)or phage (Scott and Smith, Science, 249:386-390, 1990; Devlin, Science,249:404-406, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA,87:6378-6382, 1990; and Felici, J. Mol. Biol., 222:301-310, 1991).

In one embodiment, a cell-based assay is employed in which a cell thatexpresses a RIP140 protein or biologically active portion thereof iscontacted with a test compound. The ability of the test compound tomodulate RIP140 expression or activity is then determined, e.g., bymonitoring, glucose transport, insulin sensitivity, increased energyexpenditure, increased metabolism, or increased fatty acid oxidation.The cell, for example, can be a yeast cell or a cell of mammalianorigin, e.g., rat, mouse, or human.

The ability of the test compound to modulate RIP140 binding to acompound, e.g., a RIP140 substrate, or to bind to RIP140 can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to RIP140 can bedetermined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, RIP140 can be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulateRIP140 binding to a RIP140 substrate in a complex. For example,compounds (e.g., RIP140 substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

The ability of a compound (e.g., a RIP140 substrate) to interact withRIP140 with or without the labeling of any of the interactants can beevaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with RIP140 without the labeling of either thecompound or the RIP140 (McConnell et al., Science 257:1906-1912, 1992).As used herein, a “microphysiometer” (e.g., Cytosensor®) is ananalytical instrument that measures the rate at which a cell acidifiesits environment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and RIP140.

In yet another embodiment, a cell-free assay is provided in which aRIP140 protein or biologically active portion thereof is contacted witha test compound and the ability of the test compound to bind to theRIP140 protein or biologically active portion thereof is evaluated. Ingeneral, biologically active portions of the RIP140 proteins to be usedin assays described herein include fragments that participate ininteractions with non-RIP140 molecules, e.g., fragments with highsurface probability scores.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, the ability of the RIP140 protein to bind to atarget molecule (e.g., a PPAR) can be determined using real-timeBiomolecular Interaction Analysis (BIA) (e.g., Sjolander et al., Anal.Chem., 63:2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol.,5:699-705, 1995). “Surface plasmon resonance” or “BIA” detectsbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the mass at the binding surface(indicative of a binding event) result in alterations of the refractiveindex of light near the surface (the optical phenomenon of surfaceplasmon resonance (SPR)), resulting in a detectable signal which can beused as an indication of real-time reactions between biologicalmolecules.

In various of these assays, the target gene product (RIP140) or the testsubstance is anchored onto a solid phase. The target gene product/testcompound complexes anchored on the solid phase can be detected at theend of the reaction. Generally, the target gene product is anchored ontoa solid surface, and the test compound (which is not anchored) can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either RIP140, an anti-RIP140antibody, or its target molecule (e.g., a PPAR such as PPARalpha,PPARdelta, or PPARgamma) to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to aRIP140 protein, or interaction of a RIP140 protein with a targetmolecule in the presence and absence of a test compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided that adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/RIP140fusion proteins or glutathione-S-transferase/target fusion proteins canbe adsorbed onto glutathione Sepharose™ beads (Sigma Chemical, St.Louis, Mo.) or glutathione derivatized microtiter plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or RIP140 protein. The mixture is thenincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtiter plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, and the complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of RIP140 binding or activity determined using standardtechniques.

Other techniques for immobilizing either a RIP140 protein or a targetmolecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated RIP140 protein or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

To conduct the assay, the non-immobilized component is added to thecoated surface containing the anchored component. After the reaction iscomplete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The complexes anchored on the solid surface can bedetected in a number of ways. Where the previously non-immobilizedcomponent is pre-labeled, the presence of a label immobilized on thesurface indicates that complexes were formed. Where the previouslynon-immobilized component is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface; e.g., using a labeledantibody specific for the immobilized component (the antibody, in turn,can be directly labeled or indirectly labeled with, e.g., a labeledanti-Ig antibody).

In some cases, the assay is performed utilizing antibodies reactive withRIP140 protein or target molecules, but which do not interfere withbinding of the RIP140 protein to its target molecule (e.g., a PPAR).Such antibodies can be derivatized to the wells of the plate, andunbound target or RIP140 protein trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the RIP140protein or target molecule, as well as enzyme-linked assays which relyon detecting an enzymatic activity associated with the RIP140 protein ortarget molecule.

Alternatively, cell-free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas andMinton, Trends Biochem. Sci., 18:284-7, 1993); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(e.g., Ausubel et al., eds. Current Protocols in Molecular Biology 1999,J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubelet al., eds., 1999, Current Protocols in Molecular Biology, J. Wiley:New York). Such resins and chromatographic techniques are known to oneskilled in the art (e.g., Heegaard, J. Mol. Recognit., 11:141-148, 1998;Hage et al., J. Chromatogr. B. Biomed. Sci. Appl., 699:499-525, 1997).Further, fluorescence energy transfer may also be conveniently utilized,as described herein, to detect binding without further purification ofthe complex from solution.

The assay can include contacting the RIP140 protein or a biologicallyactive portion thereof with a known compound that binds to RIP140 toform an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a RIP140 protein, wherein determining the ability of the testcompound to interact with a RIP140 protein includes determining theability of the test compound to preferentially bind to RIP140 orbiologically active portion thereof, or to modulate the activity of atarget molecule, as compared to the known compound.

A RIP140 can, in vivo, interact with one or more cellular orextracellular macromolecules, such as proteins (e.g., a PPAR). For thepurposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions are useful for regulating the activity ofthe target gene product. Such compounds can include, but are notlimited, to molecules such as antibodies, peptides, and small molecules.In general, target genes/products for use in identifying agents thatdisrupt interactions are the RIP140 genes/products identified herein. Inalternative embodiments, the invention provides methods for determiningthe ability of the test compound to modulate the activity of a RIP140protein through modulation of the activity of a downstream effector of aRIP140 target molecule. For example, the activity of the effectormolecule on an appropriate target can be determined, or the binding ofthe effector to an appropriate target can be determined, as describedherein.

To identify compounds that interfere with the interaction between thetarget gene product (a RIP140) and its binding partner(s), a reactionmixture containing the target gene product and the binding partner isprepared, under conditions and for a time sufficient, to allow the twoproducts to form a complex. To test an inhibitory agent, the reactionmixture is provided in the presence (test sample) and absence (controlsample) of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a control compound. The formation of complexesbetween the target gene product and the cellular or extracellularbinding partner is then detected. The formation of a complex in thecontrol reaction, and less formation of complex in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the target gene product and the interactivebinding partner. Such compounds are candidate compounds for inhibitingthe expression or activity or a RIP140. Additionally, complex formationwithin reaction mixtures containing the test compound and normal targetgene product can also be compared to complex formation within reactionmixtures containing the test compound and mutant target gene product.This comparison can be important in those cases wherein it is desirableto identify compounds that disrupt interactions of mutant but not normaltarget gene products.

Binding assays can be carried out in a liquid phase or in heterogenousformats. In one type of heterogeneous assay system, either the targetgene product or the interactive cellular or extracellular bindingpartner, is anchored onto a solid surface (e.g., a microtiter plate),while the non-anchored species is labeled, either directly orindirectly. The anchored species can be immobilized by non-covalent orcovalent attachments. Alternatively, an immobilized antibody specificfor the species to be anchored can be used to anchor the species to thesolid surface.

To conduct the assay, the partner of the immobilized species is exposedto the coated surface with or without the test compound. After thereaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

In another embodiment, modulators of RIP140 expression (RNA or protein)are identified. For example, a cell or cell-free mixture is contactedwith a test compound and the expression of RIP140 mRNA or proteinevaluated relative to the level of expression of RIP140 mRNA or proteinin the absence of the test compound. When expression of RIP140 mRNA orprotein is greater in the presence of the test compound than in itsabsence, the test compound is identified as a stimulator (candidatecompound) of RIP140 mRNA or protein expression. Alternatively, whenexpression of RIP140 mRNA or protein is less (statisticallysignificantly less) in the presence of the test compound than in itsabsence, the test compound is identified as an inhibitor (candidatecompound) of RIP140 mRNA or protein expression. The level of RIP140 mRNAor protein expression can be determined by methods described herein andmethods known in the art such as Northern blot or Western blot fordetecting RIP140 mRNA or protein.

In another aspect, the new methods described herein pertain to acombination of two or more of the assays described herein. For example,a modulating agent can be identified using a cell-based or a cell-freeassay, and the ability of the agent to modulate the activity of a RIP140protein can be confirmed in vivo, e.g., in an animal such as an animalmodel for obesity or diabetes (e.g., type II diabetes, e.g., ob/ob mice,db/db mice; see, e.g., Sima AAF, Shafrir E. Animal Models in Diabetes: APrimer. Taylor and Francis, Publ Amsterdam, Netherlands, 2000).

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent (compound) identified asdescribed herein (e.g., a RIP140 modulating agent, an antisense RIP140nucleic acid molecule, a RIP140 siRNA, a RIP140-specific antibody, or aRIP140-binding partner) in an appropriate animal model to determine theefficacy, toxicity, side effects, or mechanism of action, of treatmentwith such an agent. Furthermore, novel agents identified by theabove-described screening assays can be used for treatments as describedherein.

Compounds that modulate RIP140 expression or activity (RIP140modulators) can be tested for their ability to affect metabolic effectsassociated with RIP140, e.g., with decreased expression or activity ofRIP140 using methods known in the art and methods described herein. Forexample, the ability of a compound to modulate glucose transport (in thepresence or absence of insulin) can be tested using an assay for2-deoxyglucose uptake as described in Frost and Lane (J. Biol. Chem.,260:2646-2652, 1985) and glucose conversion to glyceride fatty acids canbe assayed as described in DiGirolamo et al. (J. Lipid Res., 15:332-338,1974). The conversion of white fat to brown fat can be monitored asdescribed by Tiraby et al. (J. Biol. Chem., 278:33370-33376, 2003),e.g., by assaying UCP1 (uncoupling protein 1). An increase in the amountof UCP1 or other indicator of brown fat metabolism indicates that RIP140expression or activity in inhibited.

RIP140 Modulators

Methods of modulating RIP140 expression or activity can be accomplishedusing a variety of compounds including nucleic acid molecules that aretargeted to a RIP140 nucleic acid sequence or fragment thereof, or to aRIP140 polypeptide. Compounds that may be useful for inhibiting RIP140expression or activity include polynucleotides, polypeptides, smallnon-nucleic acid organic molecules, small inorganic molecules,antibodies or fragments thereof, antisense oligonucleotides, siRNAs, andribozymes. Methods of identifying such compounds are described herein.

RNA Inhibition (RNAi)

Molecules that are targeted to a RIP140 RNA are useful for the methodsdescribed herein, e.g., inhibition of RIP140 expression, e.g., fortreating type II diabetes. Examples of nucleic acids include siRNAs(e.g., GGAATGAGCTCGATTATAA (SEQ ID NO:1); GGACAAAGGTCATGAGTGA (SEQ IDNO:2), GAATAACGCTGCCACCTTT (SEQ ID NO:3), and GAAACGCGCTCACCATAAA (SEQID NO:4)). Other such molecules that function using the mechanismsassociated with RNAi can also be used including chemically modifiedsiRNAs and vector driven expression of hairpin RNA that are then cleavedto siRNA. The nucleic acid molecules or constructs that are useful asdescribed herein include dsRNA (e.g., siRNA) molecules comprising 16-30,e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in each strand, wherein one of the strands is substantiallyidentical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%)identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to atarget region in the mRNA, and the other strand is complementary to thefirst strand. The dsRNA molecules can be chemically synthesized, cantranscribed be in vitro from a DNA template, or can be transcribed invivo from, e.g., shRNA. The dsRNA molecules can be designed usingmethods known in the art, e.g., Dharmacon.com (see, siDESIGN CENTER) or“The siRNA User Guide,” available on the Internet atmpibpc.gwdg.de/abteilungen/100/105/sirna.html.

Negative control siRNAs (“scrambled”) generally have the same nucleotidecomposition as the selected siRNA, but without significant sequencecomplementarity to the appropriate genome. Such negative controls can bedesigned by randomly scrambling the nucleotide sequence of the selectedsiRNA; a homology search can be performed to ensure that the negativecontrol lacks homology to any other gene in the appropriate genome.Controls can also be designed by introducing an appropriate number ofbase mismatches into the selected siRNA sequence.

The nucleic acid compositions that are useful for the methods describedherein include both siRNA and crosslinked siRNA derivatives.Crosslinking can be used to alter the pharmacokinetics of thecomposition, for example, to increase half-life in the body. Thus, theinvention includes siRNA derivatives that include siRNA having twocomplementary strands of nucleic acid, such that the two strands arecrosslinked. For example, a 3′ OH terminus of one of the strands can bemodified, or the two strands can be crosslinked and modified at the 3′OHterminus. The siRNA derivative can contain a single crosslink (e.g., apsoralen crosslink). In some cases, the siRNA derivative has at its 3′terminus a biotin molecule (e.g., a photocleavable biotin), a peptide(e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organiccompounds (e.g., a dye such as a fluorescent dye), or dendrimer.Modifying SiRNA derivatives in this way can improve cellular uptake orenhance cellular targeting activities of the resulting siRNA derivativeas compared to the corresponding siRNA, are useful for tracing the siRNAderivative in the cell, or improve the stability of the siRNA derivativecompared to the corresponding siRNA.

The nucleic acid compositions described herein can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, e.g., a pharmacokinetic parameter suchas absorption, efficacy, bioavailability, and/or half-life. Theconjugation can be accomplished using methods known in the art, e.g.,using the methods of Lambert et al., Drug Deliv. Rev., 47, 99-112, 2001(describes nucleic acids loaded to polyalkylcyanoacrylate (PACA)nanoparticles); Fattal et al., J. Control Release, 53:137-143, 1998(describes nucleic acids bound to nanoparticles); Schwab et al., Ann.Oncol., 5 Suppl. 4:55-8, 1994 (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem., 232:404-410, 1995(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules can also be labeled using any method known inthe art; for instance, the nucleic acid compositions can be labeled witha fluorophore, e.g., Cy3, fluorescein, or rhodamine. The labeling can becarried out using a kit, e.g., the SILENCER™ siRNA labeling kit(Ambion). Additionally, the molecule can be radiolabeled, e.g., using³H, ³²P, or other appropriate isotope.

Synthetic siRNAs can be delivered into cells by cationic liposometransfection and electroporation. Sequences that are modified to improvetheir stability can be used. Such modifications can be made usingmethods known in the art (e.g., siSTABLE™, Dharmacon). Such stabilizedmolecules are particularly useful for in vivo methods such as foradministration to a subject to decrease RIP140 expression. Longer termexpression can also be achieved by delivering a vector that expressesthe siRNA molecule (or other nucleic acid) to a cell, e.g., a fat,liver, or muscle cell. Several methods for expressing siRNA duplexeswithin cells from recombinant DNA constructs allow longer-term targetgene suppression in cells, including mammalian Pol III promoter systems(e.g., H1 or U6/snRNA promoter systems (Tuschl, Nature Biotechnol.,20:440-448, 2002) capable of expressing functional double-strandedsiRNAs; (Bagella et al., J. Cell. Physiol., 177:206-213, 1998; Lee etal., Nature Biotechnol., 20:500-505, 2002; Paul et al., NatureBiotechnol., 20:505-508, 2002; Yu et al., Proc. Natl. Acad. Sci. USA,99(9):6047-6052, 2002; Sui et al., Proc. Natl. Acad. Sci. USA,99(6):5515-5520, 2002). Transcriptional termination by RNA Pol IIIoccurs at runs of four consecutive T residues in the DNA template,providing a mechanism to end the siRNA transcript at a specificsequence. The siRNA is complementary to the sequence of the target genein 5′-3′ and 3′-5′ orientations, and the two strands of the siRNA can beexpressed in the same construct or in separate constructs. HairpinsiRNAs, driven by H1 or U6 snRNA promoter and expressed in cells, caninhibit target gene expression (Bagella et al., 1998, supra; Lee et al.,2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui etal., 2002, supra). Constructs containing siRNA sequence under thecontrol of T7 promoter also make functional siRNAs when cotransfectedinto the cells with a vector expression T7 RNA polymerase (Jacque,Nature, 418:435-438, 2002).

Animal cells express a range of noncoding RNAs of approximately 22nucleotides termed micro RNA (miRNAs) and can regulate gene expressionat the post transcriptional or translational level during animaldevelopment. miRNAs are excised from an approximately 70 nucleotideprecursor RNA stem-loop. By substituting the stem sequences of the miRNAprecursor with miRNA sequence complementary to the target mRNA, a vectorconstruct that expresses the novel miRNA can be used to produce siRNAsto initiate RNAi against specific mRNA targets in mammalian cells (Zeng,Mol. Cell, 9:1327-1333, 2002). When expressed by DNA vectors containingpolymerase III promoters, micro-RNA designed hairpins can silence geneexpression (McManus, RNA 8:842-850, 2002). Viral-mediated deliverymechanisms can also be used to induce specific silencing of targetedgenes through expression of siRNA, for example, by generatingrecombinant adenoviruses harboring siRNA under RNA Pol II promotertranscription control (Xia et al., Nat Biotechnol., 20(10): 1006-10,2002).

Injection of the recombinant adenovirus vectors into transgenic miceexpressing the target genes of the siRNA results in in vivo reduction oftarget gene expression (id). In an animal model, whole-embryoelectroporation can efficiently deliver synthetic siRNA intopost-implantation mouse embryos (Calegari et al., Proc. Natl. Acad. Sci.USA, 99:14236-14240, 2002). In adult mice, efficient delivery of siRNAcan be accomplished by “high-pressure” delivery technique, a rapidinjection (within 5 seconds) of a large volume of siRNA containingsolution into animal via the tail vein (Liu, Gene Ther., 6:1258-1266,1999; McCaffrey, Nature, 418:38-39, 2002; Lewis, Nature Genetics,32:107-108, 2002). Nanoparticles and liposomes can also be used todeliver siRNA into animals. Likewise, in some embodiments, viral genedelivery, direct injection, nanoparticle particle-mediated injection, orliposome injection may be used to express siRNA in humans.

In some cases, a pool of siRNAs is used to modulate the expression ofRIP140. The pool is composed of at least 2, 3, 4, 5, 8, or 10 differentsequences targeted to RIP140.

SiRNAs or other compositions that inhibit RIP140 expression or activityare effective for ameliorating undesirable effects of a disorder relatedto glucose transport when RIP140 RNA levels are reduced by at least 25%,50%, 75%, 90%, or 95%. In some cases, it is desired that RIP140 RNAlevels be reduced by not more than 10%, 25%, 50%, or 75%. Methods ofdetermining the level of RIP140 expression can be determined usingmethods known in the art. For example, the level of RIP140 RNA can bedetermined using Northern blot detection on a sample from a cell line ora subject. Levels of RIP140 protein can also be measured using, e.g., animmunoassay method.

Antisense Nucleic Acids

Antisense nucleic acids are useful for inhibiting RIP140. Such antisensenucleic acid molecules, i.e., nucleic acid molecules whose nucleotidesequence is complementary to all or part of an mRNA encoding a RIP140.An antisense nucleic acid molecule can be antisense to all or part of anon-coding region of the coding strand of a nucleotide sequence encodinga polypeptide of the invention. The non-coding regions (“5′ and 3′untranslated regions”) are the 5′ and 3′ sequences that flank the codingregion and are not translated into amino acids.

Based upon the sequences disclosed herein, one of skill in the art caneasily choose and synthesize any of a number of appropriate antisensemolecules to target a gene described herein. For example, a “gene walk”comprising a series of oligonucleotides of 15-30 nucleotides spanningthe length of a nucleic acid (e.g., a RIP140 nucleic acid) can beprepared, followed by testing for inhibition of expression of the gene.Optionally, gaps of 5-10 nucleotides can be left between theoligonucleotides to reduce the number of oligonucleotides synthesizedand tested.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 nucleotides or more in length. An antisensenucleic acid described herein can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The new antisense nucleic acid molecules can be administered to amammal, e.g., a human patient. Alternatively, they can be generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding a selected polypeptide to thereby inhibitexpression, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarities toform a stable duplex, or, for example, in the case of an antisensenucleic acid molecule which binds to DNA duplexes, through specificinteractions in the major groove of the double helix. An example of aroute of administration of antisense nucleic acid molecules of theinvention includes direct injection at a tissue site. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies that bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. For example, to achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs can be used in which the antisense nucleic acid molecule isplaced under the control of a strong pol II or pol III promoter.

An antisense nucleic acid molecule can be an α-anomeric nucleic acidmolecule. An α-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual, β-units, the strands run parallel to each other (Gaultier et al.,Nucleic Acids Res., 15:6625-6641, 1987). The antisense nucleic acidmolecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.,Nucleic Acids Res., 15:6131-6148, 1987) or a chimeric RNA-DNA analog(Inoue et al., FEBS Lett., 215:327-330, 1987).

Antisense molecules that are complementary to all or part of a glucosetransport-related gene are also useful for assaying expression of suchgenes using hybridization methods known in the art. For example, theantisense molecule can be labeled (e.g., with a radioactive molecule)and an excess amount of the labeled antisense molecule is hybridized toan RNA sample. Unhybridized labeled antisense molecule is removed (e.g.,by washing) and the amount of hybridized antisense molecule measured.The amount of hybridized molecule is measured and used to calculate theamount of expression of the glucose transport-related gene. In general,antisense molecules used for this purpose can hybridize to a sequencefrom a glucose transport-related gene under high stringency conditionssuch as those described herein. When the RNA sample is first used tosynthesize cDNA, a sense molecule can be used. It is also possible touse a double-stranded molecule in such assays as long as thedouble-stranded molecule is adequately denatured prior to hybridization.

Ribozymes

Ribozymes that have specificity for a RIP140 nucleic acid sequence canalso be used to inhibit RIP140 expression. Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach, Nature, 334:585-591, 1988)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. Methods of designing andproducing ribozymes are known in the art (see, e.g., Scanlon, 1999,Therapeutic Applications of Ribozymes, Humana Press). A ribozyme havingspecificity for a RIP140 nucleic acid molecule or fragment thereof canbe designed based upon the nucleotide sequence of a RIP140 cDNA. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a RIP140 RNA (Cech et al. U.S.Pat. No. 4,987,071; and Cech et al., U.S. Pat. No. 5,116,742).Alternatively, an mRNA encoding a RIP140 or fragment thereof can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules (See, e.g., Bartel and Szostak, Science,261:1411-1418, 1993).

Nucleic acid molecules that form triple helical structures can also beused to modulate RIP140 expression. For example, expression of a RIP140polypeptide can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene, Anticancer Drug Des., 6(6):569-84, 1991; Helene, Ann.N.Y. Acad. Sci., 660:27-36, 1992; and Maher, Bioassays, 14(12):807-15,1992.

A nucleic acid molecule for use as described herein can be modified atthe base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of a nucleic acid can bemodified to generate peptide nucleic acids (see Hyrup et al., Bioorganic& Medicinal Chem., 4(1): 5-23, 1996). Peptide nucleic acids (PNAs) arenucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAsallows for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols, e.g., as described inHyrup et al., 1996, supra; Perry-O′Keefe et al., Proc. Natl. Acad. Sci.USA, 93: 14670-675, 1996.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup, 1996, supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O′Keefe et al., Proc. Natl.Acad. Sci. USA, 93: 14670-675, 1996).

PNAs can be modified, e.g., to enhance their stability or cellularuptake, by attaching lipophilic or other helper groups to PNA, by theformation of PNA-DNA chimeras, or by the use of liposomes or othertechniques of drug delivery known in the art. For example, PNA-DNAchimeras can be generated which may combine the advantageous propertiesof PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAseH and DNA polymerases, to interact with the DNA portion while the PNAportion would provide high binding affinity and specificity. PNA-DNAchimeras can be linked using linkers of appropriate lengths selected interms of base stacking, number of bonds between the nucleobases, andorientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup, 1996, supra, and Finn et al.,Nucleic Acids Res., 24:3357-63, 1996. For example, a DNA chain can besynthesized on a solid support using standard phosphoramidite couplingchemistry and modified nucleoside analogs. Compounds such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be usedas a link between the PNA and the 5′ end of DNA (Mag et al., NucleicAcids Res., 17:5973-88, 1989). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al., Nucleic Acids Res., 24:3357-63, 1996).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser et al., Bioorganic Med. Chem.Lett., 5:1119-11124, 1975).

A nucleic acid targeting a RIP140 nucleic acid sequence can includeappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA,86:6553-6556, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA,84:648-652, 1989; W0 88/09810) or the blood-brain barrier (see, e.g., W089/10134). In addition, oligonucleotides can be modified withhybridization-triggered cleavage agents (see, e.g., Krol et al.,Bio/Techniques, 6:958-976, 1988) or intercalating agents (see, e.g.,Zon, Pharm. Res., 5:539-549, 1988). To this end, the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, or ahybridization-triggered cleavage agent.

RIP140 Polypeptides

Isolated RIP140 polypeptides, fragments thereof, and variants thereofare provided herein. These polypeptides can be used, e.g., as immunogensto raise antibodies, in screening methods, or in methods of treatingsubjects, e.g., by administration of the polypeptides. An “isolated” or“purified” polypeptide or biologically active portion thereof issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of polypeptides in which the polypeptideof interest is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, a polypeptide thatis substantially free of cellular material includes preparations ofpolypeptides having less than about 30%, 20%, 10%, or 5% (by dry weight)of heterologous protein (also referred to herein as “contaminatingprotein”). In general, when the polypeptide or biologically activeportion thereof is recombinantly produced, it is also substantially freeof culture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. In general, whenthe polypeptide is produced by chemical synthesis, it is substantiallyfree of chemical precursors or other chemicals, i.e., it is separatedfrom chemical precursors or other chemicals that are involved in thesynthesis of the polypeptide. Accordingly such preparations of thepolypeptide have less than about 30%, 20%, 10%, or 5% (by dry weight) ofchemical precursors or compounds other than the polypeptide of interest.

Expression of polypeptides can be assayed to determine the amount ofexpression. Methods for assaying protein expression are known in the artand include Western blot, immunoprecipitation, and radioimmunoassay.

As used herein, a “biologically active portion” of a RIP140 proteinincludes a fragment of a RIP140 protein that participates in aninteraction between a RIP140 molecule and a non-RIP140 molecule (e.g., aPPAR). Biologically active portions of a RIP140 protein include peptidesincluding amino acid sequences sufficiently homologous to the amino acidsequence of a RIP140 protein that includes fewer amino acids than afull-length RIP140 protein, and exhibits at least one activity of aRIP140 protein. Typically, biologically active portions include a domainor motif with at least one activity of the RIP140 protein (e.g., anLXXLL motif, or a PXDLS motif). A biologically active portion of aRIP140 protein can be a polypeptide that is, for example, 10, 25, 50,100, 200 or more amino acids in length. Biologically active portions ofa RIP140 protein can be used as targets for developing agents thatmodulate a RIP140 mediated activity, e.g., compounds that inhibit RIP140activity and result in at least one of increased insulin sensitivity,increased glucose transport, increased energy expenditure, increasedmetabolism, or increased fatty acid oxidation.

In some embodiments, the RIP140 polypeptide has a sequence identical toa sequence disclosed herein (e.g., a human RIP140 amino acid sequencefound under GenBank® Acc. No. Accession No. NP_(—)003480). Other usefulpolypeptides are substantially identical (e.g., at least about 45%, 55%,65%, 75%, 85%, 95%, or 99%) to the sequence found under Accession No.NP_(—)003480 and (a) retains the functional activity of RIP140 yetdiffers in amino acid sequence due to natural allelic variation ormutagenesis, or (b) exhibit an altered functional activity (e.g., as adominant negative) where desired. Provided herein are variants that havean altered amino acid sequence which can function as either agonists(mimetics) or as antagonists. Variants can be generated by mutagenesis,e.g., discrete point mutation or truncation. An agonist can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the polypeptide. An antagonist of apolypeptide can inhibit one or more of the activities of the naturallyoccurring form of the polypeptide by, for example, competitively bindingto a downstream or upstream member of a cellular signaling cascade thatincludes the polypeptide. Thus, specific biological effects can beelicited by treatment with a variant of limited function. Treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the polypeptide can have fewer sideeffects in a subject relative to treatment with the naturally occurringform of the polypeptide. In some embodiments, the variant RIP140polypeptide is a dominant negative form of RIP140. Dominant negativesare desired, e.g., in methods in which inhibition of RIP140 action isdesired, e.g., to achieve inhibition of glucose transport and/ortreatment of a metabolic disorder such as diabetes.

Also provided herein are chimeric or fusion proteins.

The comparison of sequences and determination of percent identitybetween two sequences is accomplished using a mathematical algorithm.The percent identity between two amino acid sequences is determinedusing the Needleman and Wunsch, J. Mol. Biol., 48:444-453, 1970)algorithm, which has been incorporated into the GAP program in the GCGsoftware package (available on the Internet at gcg.com), using either aBlossum 62 matrix or a PAM250 matrix, and a gap weight of 16 and alength weight of 1. The percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (also available on the Internet at gcg.com), using aNWSgapdna.CMP matrix, a gap weight of 40, and a length weight of 1.

In general, percent identity between amino acid sequences referred toherein is determined using the BLAST 2.0 program, which is available tothe public on the Internet at ncbi.nlm.nih.gov/BLAST. Sequencecomparison is performed using an ungapped alignment and using thedefault parameters (Blossum 62 matrix, gap existence cost of 11, perresidue gap cost of 1, and a lambda ratio of 0.85). The mathematicalalgorithm used in BLAST programs is described in Altschul et al.,Nucleic Acids Research 25:3389-3402, 1997.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a RIP140 protein isgenerally replaced with another amino acid residue from the same sidechain family. Alternatively, mutations can be introduced randomly alongall or part of a RIP140 coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for RIP140biological activity to identify mutants that retain activity. Theencoded protein can be expressed recombinantly and the activity of theprotein can be determined.

Antibodies

A RIP140 polypeptide, or a fragment thereof, can be used as an immunogento generate antibodies using standard techniques for polyclonal andmonoclonal antibody preparation. The full-length polypeptide or proteincan be used or, alternatively, antigenic peptide fragments can be usedas immunogens. The antigenic peptide of a protein comprises at least 8(e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acidsequence of a RIP140 polypeptide, and encompasses an epitope of RIP140such that an antibody raised against the peptide forms a specific immunecomplex with the polypeptide.

An immunogen typically is used to prepare antibodies by immunizing asuitable subject (e.g., rabbit, goat, mouse or other mammal). Anappropriate immunogenic preparation can contain, for example, arecombinantly expressed or a chemically synthesized polypeptide. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a RIP140 polypeptide as an immunogen. The antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody molecules can beisolated from the mammal (e.g., from the blood) and further purified bywell-known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when thespecific antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein, Nature, 256:495-497, 1975, the human Bcell hybridoma technique (Kozbor et al., Immunol. Today, 4:72, 1983),the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Current Protocols in Immunology, 1994, Coligan et al. (eds.)John Wiley & Sons, Inc., New York, N.Y.). Hybridoma cells producing amonoclonal antibody are detected by screening the hybridoma culturesupernatants for antibodies that bind the polypeptide of interest, e.g.,using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal antibody directed against a polypeptide can be identifiedand isolated by screening a recombinant combinatorial immunoglobulinlibrary (e.g., an antibody phage display library) with the polypeptideof interest. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; Fuchs et al.,Bio/Technology, 9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas,3:81-85, 1992; Huse et al., Science, 246:1275-1281, 1989; Griffiths etal., EMBO J., 12:725-734, 1993.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, including both human and non-human portions,which can be made using standard recombinant DNA techniques, areprovided herein. Such chimeric and humanized monoclonal antibodies canbe produced by recombinant DNA techniques known in the art, for exampleusing methods described in WO 87/02671; European Patent Application184,187; European Patent Application 171,496; European PatentApplication 173,494; WO 86/01533; U.S. Pat. No. 4,816,567; EuropeanPatent Application 125,023; Better et al., Science, 240:1041-1043, 1988;Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987; Liu et al.,J. Immunol., 139:3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci.USA, 84:214-218, 1987; Nishimura et al., Canc. Res., 47:999-1005, 1987;Wood et al., Nature, 314:446-449, 1985; and Shaw et al., J. Natl. CancerInst., 80:1553-1559, 1988); Morrison, Science, 229:1202-1207, 1985; Oiet al., Bio/Techniques, 4:214, 1986; U.S. Pat. No. 5,225,539; Jones etal., Nature, 321:552-525, 1986; Verhoeyan et al., Science, 239:1534,1988; and Beidler et al., J. Immunol., 141:4053-4060, 1988.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide. Monoclonal antibodies directed against the antigen can beobtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (Int. Rev. Immunol., 13:65-93, 1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and5,545,806. In addition, companies such as Abgenix, Inc. (Freemont,Calif.), can be engaged to provide human antibodies directed against aselected antigen using technology similar to that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Biotechnology,12:899-903, 1994).

An antibody directed against RIP140 can be used to detect thepolypeptide (e.g., in a cellular lysate or cell supernatant) to evaluateits abundance and pattern of expression. The antibodies can also be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., for example, to determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Pharmaceutical Compositions

A test compound that has been screened by a method described herein anddetermined to modulate RIP140 expression or activity, can be considereda candidate compound. A candidate compound that has been screened, e.g.,in an in vivo model of a diabetes or obesity, and determined to have adesirable effect on the disorder, e.g., by increasing glucose transport,reducing glucose levels in vivo, or reducing insulin levels, can beconsidered a candidate therapeutic agent. Candidate therapeutic agents,once screened in a clinical setting, are therapeutic agents. Candidatetherapeutic agents and therapeutic agents can be optionally optimizedand/or derivatized, and formulated with physiologically acceptableexcipients to form pharmaceutical compositions.

The compounds described herein that can modulate RIP140 expression oractivity (e.g., can modulate the interaction between RIP140 and a PPAR)can be incorporated into pharmaceutical compositions. Such compositionstypically include the compound and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. A parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation can includevacuum drying or freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. Dosage units can also be accompanied byinstructions for use.

Toxicity and therapeutic efficacy of such compounds can be determinedknown pharmaceutical procedures in cell cultures (e.g., in cultures offat cells, muscle cells, or liver cells) or experimental animals (animalmodels of obesity or of diabetes (e.g., type II diabetes). Theseprocedures can be used, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Compounds that exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in to minimize potential damageto uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies generally within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compound usedas described herein (e.g., for treating diabetes in a subject), thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, about 0.01 to 25 mg/kg body weight, about 0.1 to 20mg/kg body weight, about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can beadministered one time per week for between about 1 to 10 weeks,generally between 2 to 8 weeks, between about 3 to 7 weeks, or for about4, 5, or 6 weeks. One in the art will appreciate that certain factorsmay influence the dosage and timing required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or can include a series oftreatments. In the case of a subject suffering from diabetes, bloodglucose levels can be monitored and the dosages adjusted accordingly.

For antibodies or a fragment thereof, the dosage is about 0.1 mg/kg ofbody weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to actin the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible with such species-matched antibodies. Modifications such aslipidation can be used to stabilize antibodies and to enhance uptake andtissue penetration (e.g., into the brain). A method for lipidation ofantibodies is described by Cruikshank et al. (J. Acquired ImmuneDeficiency Syndromes and Human Retrovirology, 14:193, 1997).

Compounds that modulate expression or activity of a RIP140 are describedherein. Such a compound can be a small molecule. For example, such smallmolecules include, but are not limited to, peptides, peptidomimetics(e.g., peptoids), amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) to modulate expressionor activity of a polypeptide or nucleic acid of the invention, aphysician, veterinarian, or researcher may, for example, prescribe arelatively low dose at first, subsequently increasing the dose until anappropriate response is obtained (e.g., an appropriate blood glucoselevel). In addition, it is understood that the specific dose level forany particular animal subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

An antibody (or fragment thereof) can be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent, or a radioactive metalion. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

A nucleic acid molecule that is useful for modulating RIP140 expressionor activity can be inserted into a vector and the resulting vector usedas gene therapy vector. Gene therapy vectors can be delivered to asubject by, for example, intravenous injection, local administration(see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g.,Chen et al. (Proc. Natl. Acad. Sci. USA, 91:3054-3057, 1994). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

Compounds described herein and those identified as described herein canbe used to treat a subject that is at risk for or has a glucosetransport-related disorder such as type II diabetes. Methods ofidentifying such individuals are known in the art. Thus, methods andcompositions for both prophylactic and therapeutic methods of treating asubject at risk of (or susceptible to) a disorder or having a disorderassociated with aberrant or unwanted RIP140 expression or activity aredescribed herein. As used herein, the term “treatment” is defined as theapplication or administration of a therapeutic compound to a patient, orapplication or administration of a therapeutic compound to an isolatedtissue or cell line from a patient, who has a disease, a symptom ofdisease or a predisposition toward a disease, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease, the symptoms of disease or the predisposition towarddisease. A therapeutic compound includes, but is not limited to, smallmolecules such as small non-nucleic acid organic molecules, smallinorganic molecules, peptides, synthetic peptides, antibodies, naturalnucleic acid molecules (such as ribozymes, siRNAs, and antisenseoligonucleotides), and molecules containing nucleic acid analogs.

Provided herein are methods for preventing in a subject (e.g., a human),a disease or condition associated with an aberrant or unwanted RIP140expression or activity, by administering to the subject a RIP140 or ancompound that modulates RIP140 expression or at least one RIP140activity (e.g., RIP140 interaction with a PPAR such as PPARgamma).Subjects at risk for a disease that is caused or contributed to byaberrant or unwanted RIP140 expression or activity can be identified by,for example, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic compound can occurprior to the manifestation of symptoms characteristic of full-blowndisease, e.g., a subject exhibiting hyperglycemia but that does notexhibit effects of diabetes associated with advanced disease, such thatthe disease or disorder is prevented or, alternatively, delayed in itsprogression. Methods known in the art can be used to determine theefficacy of the treatment. The appropriate compound used for treatingthe subject can be determined based on screening assays describedherein.

It is possible that some cases of diabetes are caused, at least in part,by an abnormal level of RIP140 gene product, or by the presence of aRIP140 gene product exhibiting abnormal activity (e.g., increasedrepressor activity compared to a wild type RIP140). As such, thereduction in the level and/or activity of such gene products will bringabout the amelioration of disorder symptoms.

As discussed, successful treatment of glucose transport-relateddisorders can be brought about by techniques that serve to inhibit theexpression or activity of target gene products. For example, compounds,e.g., an agent identified using one or more of the assays describedabove, that proves to exhibit negative modulatory activity, can be usedas described herein to prevent and/or ameliorate symptoms of glucosetransport-related disorders. Such molecules can include, but are notlimited to, peptides, phosphopeptides, small organic or inorganicmolecules, or antibodies (including, for example, polyclonal,monoclonal, humanized, anti-idiotypic, chimeric or single chainantibodies, and Fab, F(ab′)₂ and Fab expression library fragments, scFVmolecules, and epitope-binding fragments thereof).

Further, siRNA, antisense, and ribozyme molecules that inhibitexpression of a RIP140 gene can also be used in accordance with themethods described herein to reduce the level of RIP140 expression, thuseffectively reducing the level of RIP140 activity. Triple helixmolecules can be utilized to reduce the level of RIP140 activity. Suchnucleic acid molecules are discussed above and in the Examples.

Another method by which nucleic acid molecules can be utilized intreating or preventing a disease that can be treated by modulatingRIP140 expression is through the use of aptamer molecules specific forRIP140 protein. Aptamers are nucleic acid molecules having a tertiarystructure that permits them to specifically bind to protein ligands(e.g., Osborne, et al., Curr. Opin. Chem. Biol., 1: 5-9, 1997; andPatel, Curr. Opin. Chem. Biol., 1:32-46, 1997). Since nucleic acidmolecules may be more conveniently introduced into target cells thantherapeutic protein molecules may be, aptamers offer a method by whichRIP140 protein activity can be specifically decreased without theintroduction of drugs or other molecules that may have pluripotenteffects.

An antibody that specifically recognizes a RIP140 can also be used.Since RIP140 is intracellular, if a whole antibody is used,internalizing antibodies are used. Lipofectin® or liposomes can be usedto deliver the antibody or a fragment of the Fab region that binds tothe RIP140 in a cell. Where fragments of the antibody are used, thesmallest inhibitory fragment that binds to the target antigen isgenerally used. For example, peptides having an amino acid sequencecorresponding to the Fv region of the antibody can be used.Alternatively, single chain neutralizing antibodies that bind tointracellular RIP140 can also be administered. Such single chainantibodies can be administered, for example, by expressing nucleotidesequences encoding single-chain antibodies within the target cellpopulation (e.g., Marasco et al., Proc. Natl. Acad. Sci. USA,90:7889-7893, 1993).

The identified compounds that inhibit RIP140 gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat, or ameliorate RIP140 disorders. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disorders.Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures as described above.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies generally within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedas described herein, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC₅₀ (i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound that is able to modulate RIP140 activity isused as a template, or “imprinting molecule,” to spatially organizepolymerizable monomers prior to their polymerization with catalyticreagents. The subsequent removal of the imprinted molecule leaves apolymer matrix that contains a repeated “negative image” of the compoundand is able to selectively rebind the molecule under biological assayconditions. A detailed review of this technique can be seen in Ansell etal., Current Opinion in Biotechnology, 7:89-94, 1996 and in Shea (Trendsin Polymer Science, 2:166-173, 1994). Such “imprinted” affinity matrixesare amenable to ligand-binding assays, whereby the immobilizedmonoclonal antibody component is replaced by an appropriately imprintedmatrix. An example of the use of such matrixes in this way can be seenin Vlatakis et al. (Nature, 361:645-647, 1993). Through the use ofisotope-labeling, the “free” concentration of compound that modulatesthe expression or activity of RIP140 can be readily monitored and usedin calculations of IC₅₀.

Such “imprinted” affinity matrixes can also be designed to includefluorescent groups whose photon-emitting properties measurably changeupon local and selective binding of target compound. These changes canbe readily assayed in real time using appropriate fiberoptic devices, inturn allowing the dose in a test subject to be quickly optimized basedon its individual IC₅₀. An rudimentary example of such a “biosensor” isdiscussed in Kriz et al., Analytical Chemistry, 67:2142-2144, 1995.

RIP140 expression or activity can be modulated for therapeutic purposes.Accordingly, in an exemplary embodiment, the modulatory methodsdescribed herein involve contacting a cell with a compound thatmodulates one or more of the activities of RIP140 protein activity(e.g., RIP140 binding to a PPAR), associated with the cell. A compoundthat modulates RIP140 activity can be a compound as described herein,such as a nucleic acid or a protein, a naturally-occurring targetmolecule of a RIP140 protein (e.g., a RIP140 substrate or receptor), aRIP140 antibody, a RIP140 agonist or antagonist, a peptidomimetic of aRIP140 agonist or antagonist, or other small molecule.

In one embodiment, the compound stimulates one or more RIP140activities. Examples of such stimulatory compounds include active RIP140protein and a nucleic acid molecule encoding RIP140. In anotherembodiment, the compound inhibits one or more RIP140 activities.Examples of such inhibitory compounds include antisense RIP140 nucleicacid molecules, antiRIP140 antibodies, and RIP140 inhibitors. Thesemodulatory methods can be performed in vitro (e.g., by culturing cellswith the compound and returning the cells to a subject) or,alternatively, in vivo (e.g., by administering the compound to asubject). As such, the new methods include treating an individualafflicted with a disease or disorder characterized by aberrant orunwanted expression or activity of a RIP140 protein or nucleic acidmolecule (e.g., type II diabetes). In one embodiment, the new methodsinvolve administering a compound (e.g., a compound identified by ascreening assay described herein), or combination of compounds thatmodulate (e.g., up regulates or down regulates) RIP140 expression oractivity. In another embodiment, the methods involve administering aRIP140 protein or nucleic acid molecule as therapy to compensate forreduced, aberrant, or unwanted RIP140 expression or activity.

Stimulation of RIP140 activity is desirable in situations in whichRIP140 is abnormally downregulated and/or in which increased RIP140activity is likely to have a beneficial effect. For example, stimulationof RIP140 activity is desirable in situations in which a RIP140 isdownregulated and/or in which increased RIP140 activity is likely tohave a beneficial effect. Likewise, inhibition of RIP140 activity isdesirable in situations in which RIP140 is abnormally upregulated and/orin which decreased RIP140 activity is likely to have a beneficialeffect.

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

EXAMPLES Example 1 Knockdown of RIP140 Potentiates Insulin-StimulatedPhosphorylation of Akt Protein Kinase

RIP140 siRNA was selected as a candidate for regulation of glucosemetabolism based on a screen using different sets of sequences expressedin mice having different metabolic profiles related to glucosemetabolism (FIG. 5).

To test the role of RIP140 in insulin-related cellular responses, siRNAswere transfected into cultured adipose cells (differentiated 3T3-L1adipocytes). siRNAs that were scrambled (nonsense), targeted to Akt,targeted to PTEN, or targeted to RIP140 were introduced into culturedcells. SiRNAs were obtained from Dharmacon. A mixture of siRNAs was usedfor the RIP140 knockdown. The mixture included the following sequencesthat are based on the RIP140 sequence found under GenBank® Acc. No.

NM_173440: GGAATGAGCTCGATTATAA; (SEQ ID NO: 1) GGACAAAGGTCATGAGTGA,(SEQ ID NO: 2) GAATAACGCTGCCACCTTT, (SEQ ID NO: 3) andGAAACGCGCTCACCATAAA. (SEQ ID NO: 4)

Differentiated 3T3-L1 adipocyte cultures (day 4 or 5) having a minimumof 90% adipocytes were transfected with 6 nmol siRNA (experimental) per150 μl cells (9×10⁶ cells/ml). Cells transfected with Akt weretransfected with 30 nmol Akt2B and 20 nmol Akt1B siRNAs. Cellstransfected with PTEN were transfected with 30 nm siRNA. Transfectionwas performed using electroporation.

Briefly, differentiated 3T3-L1 adipocytes (day 4 or 5) were transfectedwith siRNAs and plated. Cells were washed in PBS, 0.5% fat cell BSA inDMEM was added, and the cells were serum starved overnight. Insulin wasthen added to the plates (0 nM, 1.0 nM, or 100 nM in 0.5% BSA in DMEM)for 30 minutes then assayed for phosphorylation of the protein kinaseAkt using an enzyme-linked immunosorbant assay. The level of Aktphosphorylation is expressed as absorbance an OD₄₉₀.

Insulin increased the amount of Akt phosphorylation. Knockdown of RIP140increased the amount of Akt phosphorylation in the absence of insulinand increased the level of Akt phosphorylation in the presence ofinsulin over the level observed in control, insulin-treated cells (FIG.2). PTEN knockdown served as a positive control since inhibition of PTENresults in the constitutive activation of the Akt pathway.

These data demonstrate that inhibition of RIP140 (e.g., inhibition ofexpression using siRNA) can increase the cellular response to insulin.Thus, inhibition of RIP140 is useful for increasing glucose transportand is useful for treating disorders in which it is desirable toincrease such transport (e.g., type II diabetes).

Example 2 Knockdown of RIP140 Potentiates Insulin Action on DeoxyglucoseTransport Into Cultured Fat Cells

To further examine the role of RIP140 in glucose transport, the effectof RIP140 inhibition on deoxyglucose transport was assayed. Briefly,differentiated 3T3-L1 adipocytes were seeded at 150,000 cells per wellin 24 well plates and then siRNA targeted to PTEN, RIP or scrambled(control) was introduced as described above. Insulin was then added tothe plates (0 nM, 0.1 nM, or 100 nM) for 30 minutes. Cells were thenassayed for 2-deoxyglucose uptake as described in Frost and Lane (J.Biol. Chem., 260:2646-2652, 1985).

It was observed that knockdown of RIP140 increased the amount ofdeoxyglucose transport in insulin treated cells to an even greaterextent than in cells than in control cells incubated in the sameconcentration of insulin (FIG. 3).

Additional experiments were performed assaying deoxyglucose in culturedadipose cells using siRNA targeted to Anax8 (annexin A8), Aqp7(aquaporin 7), TUG, Smpd1 (sphingomyelin phosphodiesterase 1), Nrip1(RIP140), PTEN, Akt, and scrambled (FIG. 4) in the presence or absenceof insulin. These experiments further demonstrate the large increase indeoxyglucose uptake in adipose cells when RIP140 is inhibited comparedto the level of increase in during inhibition of the other sequences. Asabove, the increase in deoxyglucose uptake is even greater when RIP140is inhibited than in the positive control (PTEN).

These data further demonstrate that RIP140 can affect glucose transportand that inhibition of RIP140 is an effective method of increasingglucose transport and related metabolic activity.

Example 3 RIP140 Depletion Selectively Enhances GLUT4 Expression

The data described in Example 2 show that RIP140 is a negative regulatorof glucose uptake in 3T3-L1 cells. The enhanced glucose uptake observedwhen RIP140 is inhibited could be the result of increased insulinsignaling, enhanced GLUT4 expression, or altered GLUT4 trafficking. Wefound that RIP140 depletion significantly enhanced GLTU4 expression(FIG. 6). No change in GLUT1 expression was detected. Interestingly,knock-down of RIP140 decreased adiponectin expression, indicating thatthe change in GLUT4 expression is the result of a specific regulation byRIP140. RIP140 inhibition had no effect on actin expression. Effectivedepletion of RIP140 by an individual siRNA was confirmed using real-timePCR of in vitro transcribed message, as no effective antibody iscurrently available (FIG. 7).

We next investigated whether the RIP140 regulation of glucose uptake wasdependent upon adipogenesis. As seen in FIG. 7, RIP140 siRNA effectivelydepletes its mRNA in cells electroporated on day 4 or day 8post-differentiation. We found that RIP140 depletion effectivelyenhances glucose uptake in cells electroporated on day 4 or day 8post-differentiation (FIG. 8). This is important because GLUT4 proteinexpression in 3T3-L1 cells becomes apparent on day 4 and increases untilday 9, when its expression remains steady. Thus the absence of RIP140 isnot altering the adipogenic program to change GLUT4 expression, butregulates GLUT4 expression independently of this process.

Example 4 RIP140 Regulation of GLUT4 Expression is Independent of PPARγ

The enhancement in glucose uptake seen with RIP140 depletion isreminiscent of the increased insulin sensitivity and glucose uptake withrosiglitazone treatment, a peroxisome proliferator-activated receptor γ(PPARγ) agonist. To investigate whether RIP140 regulates glucose uptakeby modulating PPARγ activity, we used siRNA to deplete RIP140, PPARγ, orRIP140 plus PPARγ. We used day 8 adipocytes to avoid complicationsresulting from changes in adipogenesis, as PPARγ is an importantregulator of this process. Depletion of RIP140 enhanced, while PPARγdepletion inhibited, GLUT4 expression and glucose uptake (FIG. 9). Thereduction in GLUT4 expression with PPARγ knock-down was not unexpectedas it is required for the maintenance of the adipocyte phenotype (Tamoriet al., Diabetes, 51(7): 2045-55, 2002). Co-depletion of PPARγ andRIP140 resulted in an enhancement of GLUT4 expression compared to PPARγdepletion alone. This suggests that RIP140 is able to regulate GLUT4expression and thus glucose uptake in the absence of PPARγ.

Example 5 Effect of RIP140 Depletion on Adipocyte Gene Expression

We further analyzed how RIP140 depletion affects adipocyte biology by ananalysis of gene expression changes upon RIP140 depletion by Affymetrixgene chip expression profiling. Day 8 3T3-L1 adipocytes were transfectedwith scrambled or RIP140 siRNA and, after 72 hours of expression, mRNAwas harvested and used for Affymetrix gene chip analysis. Day 8 cellswere again used to avoid any complications resulting from anyalterations in adipogenesis. As shown in FIG. 10, the expression of 2747genes changes significantly upon depletion of RIP140 (of these, 40%increased and 60% decreased). The changes in gene expression were notdistributed randomly, but affected several metabolic pathways, includingglycolysis, fatty acid oxidation (FAO), the TCA cycle, and oxidativephosphorylation. The changes in individual gene expression were usuallysmall, on the order of 1.2-1.3 fold, but many genes in each pathway wereupregulated. We also found that many mitochondrial genes wereupregulated and preliminary data suggests that there may be an increasein the total number of mitochondria in RIP140-depleted cells (data notshown).

Expression of the following genes was increased significantly: C/EBPbeta, C/EBP zeta, GA repeat binding protein alpha, LXR beta, EAR2,Nur77, nuclear receptor binding factor 1, nuclear receptor interactingprotein 3, PPAR alpha, PPAR binding protein, PPAR gamma, coactivator 1beta. Expression of the following genes was decreased significantly:C/EBP delta, C/EBP gamma, Rev-ErbA alpha, LXR alpha, COUP/TFII beta,NRIP1 (i.e., RIP140), progesterone receptor membrane component 1,progesterone receptor membrane component 2, and retinoid receptor beta.Modulation of the expression of these genes, or of the activity ofpolypeptides encoded by these genes, can be used in methods to regulateglucose transport and in methods of identifying agents useful fortreatment of disorders related to glucose metabolism (e.g., diabetes,obesity).

The upregulation of mitochondrial genes suggested a change inmitochondrial number and/or function. Several transcription factors andcoregulators are thought to be important in regulating mitochondrialbiogenesis. These include the nuclear respiratory factors 1 (Nrf1) and 2(Gabp), the PPARγ coactivator α (PGC-1α)(Kelly and Scarpulla, GenesDev., 18(4):357-68, 2004), and the nuclear hormone receptors estrogenrelated receptor (Errα) (Schreiber et al., Proc Natl Acad Sci USA,101(17):6472-7, 2004; Mootha et al., Proc Natl Acad Sci USA,101(17):6570-5, 2004) and thyroid hormone receptor (TR)(Weitzel et al.,Exp. Physiol., 88(1):121-8, 2003). Although we did not see a change inthe expression of PGC-1α, which has been shown to regulate mitochondrialbiogenesis in brown adipose tissue, we did see an increase in theexpression of the related PPARγ coactivator PGC-1β and in Errα and Gabpa(FIG. 6). The increases in these transcription factors upon RIP140depletion may be sufficient to increase the transcription ofmitochondrial genes, suggesting that RIP140 is a negative regulator ofmitochondrial biogenesis.

Changes in mitochondrial gene expression are also seen in white adiposetissue during adipogenesis (Wilson-Fritch et al., Mol. Cell. Biol.,23(3):1085-94, 2003) and upon the rosiglitazone treatment of ob/ob mice(Wilson-Fritch et al., J. Clin. Invest., 114(9):1281-9, 2004).Adipogenesis and rosiglitazone treatment both enhance insulinsensitivity, as does RIP140 depletion in 3T3-L1 adipocytes.

Example 6 Evaluating RIP140 siRNA Agents in an Animal Model

Agents that inhibit expression or activity of RIP140 in vitro arefurther tested in vivo in animal models. For example, scrambled siRNA orRIP140 siRNA including SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:4 are administered to ob/ob mice using hydrodynamic transfection aspreviously described (McCaffrey, 2002, supra). Ob/ob mice can beobtained from Jackson Laboratories (Strain Name: B6.V-Lep^(ob)/J). Atvarious time points after administration of the siRNA, mRNA levels forRIP140 are measured. Additionally, the siRNA can be labeled and trackedusing methods known in the art. Levels of glucose, glucose tolerance,and plasma insulin are monitored to determine whether the RIP140 siRNAhas a beneficial effect on glucose metabolism, relative to control,i.e., whether the RIP140 siRNA causes a reduction in hyperglycemia orplasma insulin levels.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for increasing 2-deoxyglucose transport into an adipocytecell, the method comprising: providing an adipocyte cell; and contactingthe adipocyte cell with an oligonucleotide selected from the groupconsisting of an inhibitory RNA, an antisense oligonucleotide, and aribozyme, wherein said oligonucleotide inhibits RIP140 mRNA expressionin the adipocyte cell, thereby increasing 2-deoxyglucose transport intothe adipocyte cell.
 2. The method of claim 1, wherein theoligonucleotide is an inhibitory RNA.
 3. The method of claim 2, whereinthe inhibitory RNA is a small inhibitory RNA (siRNA).
 4. The method ofclaim 1, wherein the adipocyte cell is contacted in vitro.
 5. The methodof claim 1, wherein the adipocyte cell is contacted in vivo.
 6. Themethod of claim 3, wherein the siRNA comprises a sequence selected fromSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
 7. The method ofclaim 1, wherein the oligonucleotide enhances expression of GLUT4protein in the adipocyte cell.
 8. A method for increasing 2-deoxyglucosetransport into an adipocyte cell in a subject that is at risk for orsuffering from a disorder related to glucose metabolism, the methodcomprising: administering to the subject an oligonucleotide selectedfrom the group consisting of an inhibitory RNA, an antisenseoligonucleotide, and a ribozyme, wherein said oligonucleotide inhibitsRIP140 mRNA expression in an amount sufficient to increase2-deoxyglucose transport into an adipocyte cell in the subject, therebyincreasing 2-deoxyglucose transport into an adipocyte cell in thesubject.
 9. The method of claim 8, wherein the disorder is type Idiabetes, type II diabetes, or obesity.
 10. The method of claim 1,wherein the oligonucleotide is an antisense oligonucleotide.
 11. Themethod of claim 1, wherein the oligonucleotide is a ribozyme.
 12. Themethod of claim 8, wherein the oligonucleotide is an inhibitory RNA. 13.The method of claim 12, wherein the inhibitory RNA is a small inhibitoryRNA (siRNA).
 14. The method of claim 13, wherein the siRNA comprises asequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:4.
 15. The method of claim 8, wherein the oligonucleotide is anantisense oligonucleotide.
 16. The method of claim 8, wherein theoligonucleotide is a ribozyme.
 17. The method of claim 8, wherein theoligonucleotide enhances expression of GLUT4 protein in the adipocytecell.